This project is directed toward understanding the biology of gene transfer into hematopoietic progenitor and stem cells for therapeutic purposes. Our initial studies have focused on retrovirus vectors to infect autologous progenitor and stem cells. To date we have been able to demonstrate reproducible, stable gene transfer into marrow-repopulating cells. The genes are present and expressed in myeloid (granulocyte, monocyte, erythrocyte, platelet) and lymphoid (CD3, CD4, CD8, CD20 positive lymphocytes) lineages. We have developed protocols that allow gene transfer into and expression of the transferred gene by up to 20% of blood cells early after transplant and stable expression in 1% to 3% of blood cells long term. We have developed a competitive repopulation strategy that allows us to study up to 3 different vectors simultaneously in a single transplanted animal. This approach coupled with semi-quantitative PCR and flow cytometric analysis allows direct comparison of vector pseudotypes and vector constructs using a minimal number of animals. Future directions are to enhance our ability to perform quantitative comparisons of increasing numbers of vectors simultaneously, and to study non-retrovirus vectors for the ability to transduce different progenitor and stem cell populations. Collaborations have been established to study lentiviral, AAV, and SV40 based vectors. FUNDING NIH grants RR00166, AI35191, CA15704, AI37747 and HL54881 and Amgen Corp. Kiem, H.P., Andrews, R.G., Morris, J., Peterson, L., Heyward, S., Allen, J.M., Rasko, J.E., Potter, J., and Miller, A.D. Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and magakaryocyte growth and development factor. Blood 92:1878-1886, 1998. Winkler, A., Kiem, H-P., Shields, L.E., Sun, Q-H., and Andrews, R.G. Gene transfer into fetal baboon hematopoietic progenitor cells. Hum. Gene Ther. 10:667-677, 1999. Emery, D.W., Wang, X.C., Andrews, R.G., and Papayannopoulou, Th. Optimizing monkey models for bone marrow transduction with retroviral vectors. Abstr. Am. Soc. for Gene Therapy, May 1998. Andrews, R.G., Winkler, A., Shields, L.E., and Kiem, H-P. Gene transfer into fetal baboon hematopoietic progenitors. Abstr. Am. Soc. for Gene Therapy, May 1998. Rasko, J.E.J., Andrews, R.G., Morris, J.C., Gottschalk, R.J., Peterson, L.J., Potter, J., Miller, A.D., and Kiem, H-P. Temporal and lineage specific monitoring of GFP expression in following retroviral transduction of multipotential repopulating hemopoietic cells in the baboon. Abstr. Am. Soc. for Gene Therapy, May 1998. Kiem, H-P., Andrews, R.G., Morris, J.C., Heyward, S., Peterson, L.J., Potter, J., Allen, J.M., and Miller, A.D. High level gene transfer into baboon hematopoietic stem cells using recombinant human fibronectin fragment CH-296 in combination with IL-6, SCF, Flt3-L, and MGDF. Abstr. Am. Soc. for Gene Therapy, May 1998. Kiem, H-P., Rasko, J.E.J., Morris, J., Peterson, L.J., Miller, A.D., and Andrews, R.G. Engraftment kinetics of expanded and transduced baboon CD34-enriched cells selected for green fluorescent protein expression. Abstr. Am. Soc. Hematol., December 1998.